In optical scanning microscopy, a specimen is scanned with a probe by moving a specimen relative to a stationary beam or by moving a beam relative to a stationary specimen. Alternatively, a spinning disk containing an array of apertures can be placed between the light source and the specimen so that regions of the specimen are illuminated in a patterned and sequential manner. One objective of laser scanning confocal microscopy is to realize diffraction limited spatial resolution through the use of strategically placed apertures. The general properties of optical scanning microscope systems are considered in detail in: Theory and Practice of Scanning Optical Microscopy, Tony Wilson and Colin Sheppard (Academic Press, New York, 1984); Confocal Microscopy, edited by Tony Wilson (Academic Press, New York, 1990); and the Handbook of Biological Confocal Microscopy, edited by James B. Pawley (Plenum Press, New York, 1990) and articles referenced therein.
In a number of instances, such as applications involving confocal microscopy, movement of the specimen relative to a fixed illumination source has proven too slow. Therefore, it has become customary to scan a stationary specimen with an illumination light, typically a laser beam, to increase scan rates. Movement of the illumination light along X and Y axes in the plane orthogonal to the optical (Z) axis has most commonly been accomplished using mirrors displaced in space by mechanical devices, such as closed loop and resonant galvanometers. Light that interacts with the sample (by reflection, refraction, fluorescence, etc.) is collected in a serial fashion (i.e. one point at a time) and reconstructed within a computer's memory in order to form an image. The typical pattern to perform this operation is in the form of a raster pattern or 2-dimensional grid produced by rapidly scanning along a straight line in one axis (X direction), then moving the beam one grid location in the orthogonal axis (Y direction), followed by a scan along a parallel straight line in the X axis (in either the forward or reverse direction). Repeated application of this sequence allows the construction of a two-dimensional grid or raster-scanned image of a rectangular field of view. Such an image can readily be viewed on any device organized as a two-dimensional grid display such as a computer screen or television.
An inefficiency of the current technique is caused by the fact that beam-directing devices with inertia cannot instantaneously start or stop their movements. At the end of each line segment, the beam must be directed to retrace or turn around in order to initiate the scanning of a new line. During this time, no useful data are collected. This retrace or turn around time ultimately limits the temporal resolution (i.e. the number of images that can be collected per unit time). The greatest scan rates achieved with this approach have yielded acquisition rates on the order of 30 frames/sec for images containing 512×512 pixels or less. Higher rates can be achieved by decreasing the dimensions of the scanned region. However, even if spatial resolution is compromised by reducing the number of points in each dimension, turn around time severely limits the maximum temporal resolution that can be attained. The ultimate compromise in spatial resolution is the so-called “line scan” in which a beam is simply directed back and forth along a single line. Even during line scans, a significant time is spent in the “turn around” mode.
In another approach for high rate scanning confocal microscopy, acousto-optical beam deflector (AOD) devices, which do not have any moving mechanical parts, have been implemented as beam-steering devices to increase scan speeds. However, this approach is limited by the small deflection angles and the optical properties of AOD devices. In particular, light at different wavelengths is deflected at different angles. Consequently, it is difficult to illuminate the same spot in the specimen simultaneously with different wavelengths of light, and in the case of fluorescence microscopy, light coming from the specimen does not move along the same path as light directed toward the specimen after passing through the AOD. As a consequence, AOD devices are typically not placed in the path of light coming from the specimen and it is not possible to focus light emanating from the image plane in the specimen at a pinhole placed in front of the detector, as is required for true confocal microscopy. Instead, light is often passed through a slit, which permits more out-of-focus light to reach the detector. In this approach, optical performance is sacrificed for speed.
A number of applications, including the observation of any process where changes occur over the period of less than 1 second, would benefit from faster scan speeds than have been achieved to date. In addition, processes that utilize a scanned beam to affect a surface in a spatially organized manner, such as high resolution lithography or image projection systems, would benefit from faster scan rates. Therefore, there is a need to increase the rates at which specimens and/or surfaces are scanned without sacrificing imaging performance such as spatial resolution.